A Monovalent Chimpanzee Adenovirus PDF

The
n e w e ng l a n d j o u r na l
of
m e dic i n e
Original Article
A Monovalent Chimpanzee Adenovirus

Ebola Vaccine Boosted with MVA
K. Ewer, T. Rampling, N. Venkatraman, G. Bowyer, D. Wright, T. Lambe,
E.B. Imoukhuede, R. Payne, S.K. Fehling, T. Strecker, N. Biedenkopf, V. Krhling,
C.M. Tully, N.J. Edwards, E.M. Bentley, D. Samuel, G. Labb, J. Jin, M. Gibani,
A. Minhinnick, M. Wilkie, I. Poulton, N. Lella, R. Roberts, F. Hartnell, C. Bliss,
K. SierraDavidson, J. Powlson, E. Berrie, R. Tedder, F. Roman, I. DeRyck,
A. Nicosia, N.J. Sullivan, D.A. Stanley, O.T. Mbaya, J.E. Ledgerwood,
R.M. Schwartz, L. Siani, S. Colloca, A. Folgori, S. DiMarco, R. Cortese, E. Wright,
S. Becker, B.S. Graham, R.A. Koup, M.M. Levine, A. Volkmann, P. Chaplin,
A.J. Pollard, S.J. Draper, W.R. Ballou, A. Lawrie, S.C. Gilbert, and A.V.S. Hill
A BS T R AC T
BACKGROUND
The West African outbreak of Ebola virus disease that peaked in 2014 has caused more
than 11,000 deaths. The development of an effective Ebola vaccine is a priority for control
of a future outbreak.
METHODS
In this phase 1 study, we administered a single dose of the chimpanzee adenovirus 3

(ChAd3) vaccine encoding the surface glycoprotein of Zaire ebolavirus (ZEBOV) to 60
healthy adult volunteers in Oxford, United Kingdom. The vaccine was administered in
three dose levels 11010 viral particles, 2.51010 viral particles, and 51010 viral particles
with 20 participants in each group. We then assessed the effect of adding a booster
dose of a modified vaccinia Ankara (MVA) strain, encoding the same Ebola virus glycoprotein, in 30 of the 60 participants and evaluated a reduced primeboost interval in
another 16 participants. We also compared antibody responses to inactivated whole
Ebola virus virions and neutralizing antibody activity with those observed in phase 1
studies of a recombinant vesicular stomatitis virusbased vaccine expressing a ZEBOV
glycoprotein (rVSV-ZEBOV) to determine relative potency and assess durability.
The authors full names, academic de

grees, and affiliations are listed in the
Appendix. Address reprint requests to
Dr. Hill at the Jenner Institute, University
of Oxford, Old Road Campus Research
Bldg., Headington, Oxford OX3 7DQ,
United Kingdom, or at adrian.hill@ndm.ox
.ac.uk.
Drs. Ewer, Rampling, and Venkatraman
contributed equally to this article.
A preliminary version of this article
was published on January 28, 2015, at
NEJM.org.
N Engl J Med 2016;374:1635-46.
DOI: 10.1056/NEJMoa1411627
Copyright 2016 Massachusetts Medical Society.
RESULTS
No safety concerns were identified at any of the dose levels studied. Four weeks after
immunization with the ChAd3 vaccine, ZEBOV-specific antibody responses were similar
to those induced by rVSV-ZEBOV vaccination, with a geometric mean titer of 752 and 921,
respectively. ZEBOV neutralization activity was also similar with the two vaccines (geometric mean titer, 14.9 and 22.2, respectively). Boosting with the MVA vector increased
virus-specific antibodies by a factor of 12 (geometric mean titer, 9007) and increased
glycoprotein-specific CD8+ T cells by a factor of 5. Significant increases in neutralizing
antibodies were seen after boosting in all 30 participants (geometric mean titer, 139;
P<0.001). Virus-specific antibody responses in participants primed with ChAd3 remained
positive 6 months after vaccination (geometric mean titer, 758) but were significantly
higher in those who had received the MVA booster (geometric mean titer, 1750; P<0.001).
CONCLUSIONS
The ChAd3 vaccine boosted with MVA elicited B-cell and T-cell immune responses to
ZEBOV that were superior to those induced by the ChAd3 vaccine alone. (Funded by the
Wellcome Trust and others; ClinicalTrials.gov number, NCT02240875.)
n engl j med 374;17nejm.org April 28, 2016
The New England Journal of Medicine

Downloaded from nejm.org on April 28, 2016. For personal use only. No other uses without permission.
Copyright 2016 Massachusetts Medical Society. All rights reserved.
1635
The
he recent outbreak of Ebola virus

disease (EVD) in West Africa has led to
more than 11,000 deaths, with a peak in
mortality from August through December of
2014 and a subsequent decline in the number
of new cases. The development of a durable and
effective Ebola vaccine is a priority both to eliminate the remnants of the outbreak and to prevent and control future epidemics. Several candidate vaccines have shown promising results in
phase 1 trials,1-6 and a recombinant vesicular
stomatitis virusbased vaccine expressing the surface glycoprotein of Zaire ebolavirus (rVSV-ZEBOV)
showed efficacy in an interim analysis of a
phase 3 trial in Guinea (ring vaccination trial).7
More data will be required before the rVSV-ZEBOV
vaccine can be licensed. However, the use of this
vaccine could contribute to ending the current
outbreak in West Africa by limiting the spread of
infection among close contacts of persons with
EVD. In this context, the duration of vaccine efficacy can be relatively short, since the time
since exposure is typically known and protection
is conferred within the time frame necessary to
prevent clinical disease and transmission. In a
different context, during the earlier, uncontrolled
phase of an outbreak in which most transmission is undetected and new cases appear in geographically disparate locations, an effective vaccine would need to have longer durability. For
this earlier phase of the outbreak, longer-lasting
vaccine efficacy would be required to provide
sufficient protection to the entire population
within an affected area to interrupt transmission, particularly where transmission is unpredictable.
The demonstration in humans of vaccine efficacy against EVD with the rVSV-ZEBOV vaccine
has facilitated the development of an Ebola virus
vaccine by adding to our knowledge of immunity associated with protection, data that were
previously derived only from rodent and primate
challenge models. Before the current outbreak
and the subsequent trial of rVSV-ZEBOV, licensure of an Ebola vaccine was dependent on the
demonstration of adequate immunogenicity and
safety in humans, along with linkage to immunogenicity and efficacy data in challenge studies
conducted in nonhuman primates.8 Now we can
compare cellular and humoral immune responses
induced by various candidate vaccines in phase 1
1636
of
m e dic i n e
studies with responses observed in rVSV-ZEBOV

trials, in which various measures of humoral
immunity (e.g., ZEBOV glycoproteinspecific
antibody responses and neutralizing antibody
titers) have been described in African and European cohorts.4 In contrast, substantial cellular
immunogenicity induced by rVSV-ZEBOV immunization has not been shown in nonhuman primate
models or in recent human phase 1 trials.3,4,9,10
The induction of both antibodies and CD8+
T-cell responses is potentially protective against
EVD. Antibody levels as measured on an enzymelinked immunosorbent assay (ELISA) against the
Mayinga strain glycoprotein of ZEBOV had broad
correlation with protection across a range of
studies of vectored vaccination conducted in
cynomolgus macaques, with a reciprocal titer of
3700 correlating with complete protection against
challenge.11,12 However, after immunization of
macaques with a protective vaccine dose of human serotype 5 adenovirus (AdHu5), antibodies
did not adoptively transfer protection to other
macaques, and depletion of CD8+ T cells largely
ablated protection.13 This finding indicates a
potential role for induced CD8+ T cells in vaccine
efficacy and the likelihood that the observed
antibody correlate is not a causal mechanism.
Such immune activity may reflect a constellation
of induced T-cell and antibody responses, both
of which may contribute to protection. In cynomolgus macaques, the addition of a booster vaccination with a modified vaccinia Ankara (MVA)
strain to priming immunization with the chimpanzee adenovirus 3 (ChAd3) vaccine encoding
the ZEBOV surface glycoprotein increased immunogenicity by a factor of at least 10 and increased
the duration of protective efficacy against Ebola
virus challenge from 5 weeks to 10 months after
vaccination,11 which indicates that boosting improves both immunogenicity and durability of
protection.
Me thods
Study Participants
The study was conducted at the Centre for

Clinical Vaccinology and Tropical Medicine at
the University of Oxford. Participants were
healthy adults between the ages of 18 and 50
years who provided written informed consent
(Table1).

9 (90)
Female
Range yr
1 (10)

0
22.028.5
25.21.9
5 (50)
5 (50)
17.433.0
24.54.0
1 (10)
3 (30)
5 (50)
1 (10)
10 (100)
2448
35.28.2
3 (30)
3 (30)
4 (40)
2 (20)
8 (80)
Unboosted
(N=10)
24.72.9
1 (10)
3 (30)
6 (60)
1 (10)
9 (90)
2248
30.87.4
1 (10)
3 (30)
6 (60)
5 (50)
5 (50)
Boosted
(N=10)
21.430.7
Group 2
20.426.6
22.72.0
2 (20)
8 (80)
1 (10)
9 (90)
1848
31.911.2
3 (30)
2 (20)
2 (20)
3 (30)
3 (30)
7 (70)
Unboosted
(N=10)
22.82.7
1 (10)
8 (80)
1 (10)
1 (10)
9 (90)
2242
39.97.6
2 (20)
1 (10)
7 (70)
4 (40)
6 (60)
Boosted
(N=10)
17.127.4
Group 3
18.627.8
22.23.7
2 (25)
6 (75)
1 (12)
7 (88)
2150
28.010.0
1 (12)
1 (12)
6 (75)
5 (62)
3 (38)
Group 4
(N=8)
18.529.0
23.43.1
2 (25)
6 (75)
1 (12)
7 (88)
2043
27.88.1
1 (12)
1 (12)
5 (62)
1 (12)
4 (50)
4 (50)
Group 5
(N=8)
17.133.0
23.73.2
3 (4)
21 (28)
50 (66)
2 (3)
2 (3)
4 (5)
70 (92)
1850
30.98.8
15 (20)
18 (24)
39 (51)
4 (5)
37 (49)
39 (51)
All
Participants
(N=76)
* Plusminus values are means SD. There were no significant differences between the study groups. Percentages may not total 100 because of rounding.
Among the 30 participants who received a booster dose of the MVA vaccine, 18 received a dose of 1.5108 plaque-forming units (PFU) and 12 received a dose of 3108 PFU, with strati
fication according to priming-dose group.
Two additional groups of 8 participants each were recruited to assess the effect of reducing the interval between priming and boosting to either 1 week (group 4) or 2 weeks (group 5).
The 16 participants in these two groups received a priming dose of 2.51010 viral particles of ChAd3 and a boosting dose of 1.5108 PFU of MVA.
Race was self-reported.
The body-mass index is the weight in kilograms divided by the square of the height in meters.
23.84.3
18.530.5
1 (10)
30
Range
3 (30)
2529.9
Mean
6 (60)
18.524.9
<18.5
Distribution no. (%)
Body-mass index
0
Asian
Mixed
9 (90)
Black
10 (100)
2242
White
Race no. (%)
2 (20)
3 (30)
2148
4150 yr
30.48.2
2 (20)
3140 yr
5 (50)
5 (50)
5 (50)
32.210.0
4 (40)
2130 yr
Mean yr
0
4 (40)
1820 yr
Distribution no. (%)
Age
1 (10)
Boosted
(N=10)
Group 1
Unboosted
(N=10)
Male
Sex no. (%)
Characteristic
Table 1. Characteristics of the Participants at Baseline.*
A Monovalent Chimpanzee Adenovirus Ebola Vaccine
1637
The
Study Oversight
The study was reviewed and approved by the

United Kingdom National Research Ethics Service, the Committee South CentralOxford A,
the Medicines and Healthcare Products Regulatory Agency, and the Oxford University Clinical
Trials and Research Governance team, who monitored compliance with Good Clinical Practice
guidelines. An independent data and safety monitoring board provided safety oversight.
The ChAd3 vaccine was provided by the Vaccine Research Center of the National Institute of
Allergy and Infectious Diseases (NIAID) and
GlaxoSmithKline, which manufactured the vaccine. The MVA vaccine was produced under a
contract between NIAID and Fisher BioServices.
Study Design
In this phase 1 study, we administered the ChAd3

vaccine to 60 participants; 20 participants received the vaccine in a dose of 11010 viral particles (group 1), 20 received the vaccine in a dose
of 2.51010 viral particles (group 2), and 20 received the vaccine in a dose of 51010 viral particles (group 3). In addition, in an attempt to
improve immune responses, we invited 10 participants from each of the three groups to receive
a single booster dose of MVA (called MVA-BN
Filo), which encodes the same Mayinga strain
glycoprotein antigen as that encoded by the
ChAd3 vaccine, along with glycoproteins of the
Sudan Ebola virus species and Marburg virus and
the nucleoprotein of Ta Forest Ebola virus.
From late November to early December 2014
(at 3 to 10 weeks after the priming immunization), we administered the MVA vaccine at a dose
of 1.5108 plaque-forming units (PFU) to 18 participants and at a dose of 3108 PFU to 12 participants, with stratification according to primingdose group. We then recruited and immunized
two additional groups of 8 participants each to
assess the effect of reducing the interval between priming and boosting to either 1 week
(group 4) or 2 weeks (group 5). In this analysis,
all the participants received a priming dose of
2.51010 viral particles of ChAd3 and a boosting
dose of 1.5108 PFU of MVA.
Details regarding the study design and participants are provided in Figure S1 in the Supplementary Appendix, available with the full text of
this article at NEJM.org. Additional data on vaccines, safety-assessment techniques, and study
1638
of
m e dic i n e
design are provided in the study protocol, also

available at NEJM.org.
Assessment of Humoral Immunity
We assessed antibody responses using four separate types of IgG ELISA: an in-house standardized
ELISA that was developed at the Jenner Institute
and uses a recombinant ZEBOV glycoprotein, a
commercially available ZEBOV glycoprotein ELISA
kit (Alpha Diagnostic International), an end-point
ELISA performed at the National Institutes of
Health with a readout for the EC90 assay (the
concentration at which there is a 90% decrease
in antigen binding), and a whole-virion ELISA
that uses inactivated ZEBOV Makona (the current outbreak strain). Two assays were used to
measure neutralizing antibodies. The first measured direct neutralization of live ZEBOV (Mayinga
strain) from all participants who received the
booster dose at 28 days after the dose of ChAd3
vaccine and 14 days after the dose of the MVA
vaccine. The second measured the blocking ability of vaccine-induced antibodies with the use of
a pseudotyped lentivirus expressing the glycoprotein from the Mayinga strain, with a readout
of the 50% inhibitory concentration (IC50) assay.
A competitive ELISA-based assay was also used
to detect blocking of a neutralizing monoclonal
antibody (4G7)14 by serum after boosting with
MVA. (A detailed description of the immunologic analyses is provided in the Supplementary
Appendix.)
T-Cell Assays
We measured T-cell responses to vaccination using ex vivo interferon- enzyme-linked immunosorbent spot (ELISPOT) assays at all time points
and flow cytometry with intracellular cytokine
staining at the peak of the immune response after each vaccination. T-cell assays were performed
on freshly isolated peripheral-blood mononuclear
cells (PBMCs).
R e sult s
Study Population
A total of 76 of the 123 volunteers who were

screened for eligibility were vaccinated (Fig. S1 in
the Supplementary Appendix). Of the 60 participants who were included in the original analysis
of the ChAd3 vaccine, 59 completed at least 28 days
of follow-up. One participant in group 1 withdrew

on day 1 after vaccination owing to an aversion

to venipuncture. Communication with the participant on day 10 after vaccination confirmed
that the participant remained well, with no symptoms to report. Among the participants who
were followed for 180 days were all 30 who received the MVA booster and 28 who did not
receive the booster.
in group 2, and eight in group 3; moderate lymphocytopenia was noted in two participants each
in group 2 and group 3 on day 1. Transient mild
or moderate elevations in bilirubin were recorded
in three participants in group 2 and three in
group 3. Transient hyperbilirubinemia in the
severe range was recorded in two participants
(one in group 2 and one in group 3) who had a
prevaccination diagnosis of Gilberts syndrome.
Safety
A complete list of the frequency and maximum

severity of solicited, unsolicited, and laboratory
adverse events, according to dose group, is provided in Tables S1 through S8 in the Supplementary Appendix. The majority of adverse events
that were reported in all dose groups were mild
in severity, with no unexpected serious adverse
reactions or serious adverse events. Local reactogenicity appeared to be more pronounced after
boosting vaccination than after priming vaccination, a finding that is consistent with the results
of other studies of heterologous primeboost
vaccine schedules incorporating a ChAd prime
and MVA booster. In contrast, fewer systemic
adverse events were reported with boosting vaccination than with priming vaccination.
The majority of adverse events were self-limited
and mild. Local pain was the most common
local event (with one case reported as severe).
Moderate systemic adverse events were fever,
myalgia, arthralgia, headache, fatigue, nausea,
and malaise. No severe systemic solicited adverse
events were reported. Four episodes of mild fever
(37.6 to 38.0C in 4 participants) were reported.
No fever persisted for more than 24 hours.
A prolonged activated partial-thromboplastin
time was observed in four participants during
the first 2 weeks after vaccination (three with a
grade 1 elevation and one with a grade 2 elevation). None of the prolongations were associated
with symptoms or clinical features of coagulopathy. The elevations fully resolved in all participants by 10 weeks after vaccination. No further
abnormality was found on extended hematologic
and coagulation evaluation.
A transient induction of an antiphospholipid
antibody causing an in vitro artifact on the laboratory assay for activated partial-thromboplastin
time after the administration of adenovirus vectors has been reported previously.15,16 Transient
mild lymphocytopenia was noted on day 1 after
vaccination in five participants in group 1, four
Antibody Responses
Antibody responses as measured by means of

standardized glycoprotein ELISA increased significantly by 7 days after the MVA dose and
peaked at day 14 after boosting and then decreased slightly by day 28 (P<0.01 by the Friedman test for all comparisons) (Fig.1A). Responses remained significantly above pre-boost levels
at 180 days after MVA vaccination and were four
times as high as titers measured at 180 days
after priming with the ChAd3 vaccine alone
(P<0.001 by the MannWhitney test) (Fig.1B); in
addition, 100% of the participants who received
the MVA vaccine remained seropositive, as compared with less than half of those who received
the priming vaccination alone.
Titers on whole-virion ELISA showed that immunogenicity at 4 weeks after priming with
ChAd3 was similar to that measured after immunization with rVSV-ZEBOV in 10 vaccinees in
Hamburg, Germany, at the dose administered
in the ring vaccination study (geometric mean
titer with ChAd3, 752.4; 95% confidence interval
[CI], 541 to 1647; geometric mean titer with
rVSV-ZEBOV, 920.7; 95% CI, 541 to 1566). In our
study, after boosting with MVA, titers increased
by a factor of 9 (geometric mean titer, 6625; 95%
CI, 4748 to 9245) at 1 week and by a factor of 12
(geometric mean titer, 9007; 95% CI, 6909 to
11741) at 4 weeks (Fig.1C).
Six months after vaccination, titers in the
group primed only with ChAd3 were similar to
those detected 1 month after vaccination (geometric mean titer, 758; 95% CI, 561 to 1023;
P=0.90 by the two-tailed Wilcoxon matchedpairs test). Titers remained significantly higher
in the group that received the MVA booster
(geometric mean titer, 1750; 95% CI, 1247 to
2456) than in the ChAd3 prime-only group
(P<0.001 by the two-tailed MannWhitney test).
At that time, 77% of vaccinees in the group that
received the MVA booster remained seropositive,

1639
The
A Antibody Response on Standardized ELISA
of
m e dic i n e
B Antibody Response on ZEBOV GP IgG ELISA

*
ChAd3
MVA
Standardized ZEBOV GP IgG
2.51010 Viral particles
5000
51010 Viral particles
4000
3000
2000
1000
104
103
102
101
+1
80
+9
0
+2
8
+0
Prime Only
No.
Percent Positive
C Antibody Response in Whole ZEBOV Virions
18
0
90
A+
A+
A+
+1
80
M
A+
18
0
A+
A+ 0
A + 14
2
M 8
M +0
+
M 14
+2
8
Day
Day
Day 7
28
100
A+
Standardized ELISA
11010 Viral particles
29
3
29
83
29
55
PrimeBoost
29 29
38 3
30
100
30
97
30
90
D Neutralizing Antibodies against Live ZEBOV
Day 28
Day 180
-Z
rV
Vaccine Regimen (day)
as compared with 25% of those in the primeonly group. (Summary data are provided in Tables S9 through S12 in the Supplementary Appendix.)
Neutralizing antibody titers to live ZEBOV
(Mayinga strain) from all participants who received the MVA booster were measured at 28
days after the ChAd3 dose and at 14 days after
the MVA dose (Fig.1D). Low levels of neutralizing antibodies were detected in participants at
28 days (geometric mean titer, 14.9; 95% CI, 12
to 18.5) levels that were similar to those reported after the rVSV-ZEBOV vaccine4 (geometric
mean titer, 22.2; 95% CI, 15.7 to 31.4); by 14
1640
Ch
SV
Ad
Ch
Vaccine Regimen
3
(d MV
ay A
14
)
VA
M
Ad
Ch
M
Ad
VA
3
Ad
Ch
Ch
SV
rV
Ch
Ad
10
VA
102
Ad
103
Ch
(d Ad
ay 3
28
)
104
512
256
128
64
32
16
8
4
2
1
EB
(d OV
ay
28
)
Neutralizing Antibody Titer

(Mayinga)
Whole-Virion ELISA
105
days after the MVA vaccine, the levels had increased by a factor of 9 (geometric mean titer,
139; 95% CI, 90 to 215) and all participants were
seropositive (geometric mean titer, >8). Boosting with the high dose of MVA elicited neutralizing antibody titers that were higher than those
with the low dose (geometric mean titer in the
high-dose group, 243.9; 95% CI, 96 to 628; geometric mean titer in the low-dose group, 95.7;
95% CI, 65 to 142; P=0.03 by the two-tailed
MannWhitney test) (Fig.1D). (Additional details regarding neutralizing antibodies and IgG
antibodies are provided in Fig. S2 and S3 in the
Supplementary Appendix.)

Figure 1 (facing page). Antibody Responses to the Zaire

ebolavirus (ZEBOV) Glycoprotein.
Panel A shows the geometric mean titer of antibody re
sponses to increasing doses of the chimpanzee adeno
virus 3 (ChAd3) vaccine encoding the surface glyco
protein of ZEBOV, followed by a booster dose of a
modified vaccinia Ankara (MVA) strain. Antibody re
sponses are shown according to measurements on a
standardized enzyme-linked immunosorbent assay
(ELISA) for doses of 11010 viral particles (in 19 partici
pants), 2.51010 viral particles (in 20 participants), and
51010 viral particles (in 20 participants). Solid symbols
indicate that participants received only the ChAd3 vac
cine, and open symbols, that participants received the
ChAd3 vaccine followed by booster MVA. Antibody re
sponses increased significantly by 7 days after the MVA
dose and peaked at day 14 after boosting and then de
creased slightly by day 28. There were no significant
differences in responses among the dose groups in
the cohort that received the MVA booster at any time
point after vaccination. The days of the analysis are indi
cated by a plus sign after administration of the ChAd3
vaccine (A) and the MVA vaccine (M). Panel B shows
the responses of participants after administration of
the prime and booster vaccines, according to results on
anti-ZEBOV glycoprotein (GP) IgG ELISA. The solid hori
zontal lines represent the geometric mean titer. Percent
ages of vaccinees with positive antibody responses at
each time point are indicated below the graph. The hori
zontal dashed line represents the threshold for a posi
tive result (arbitrary ELISA units, +0.561), calculated as
the mean plus 3 standard deviations of the response on
day 0 for all participants. Panel C shows antibody titers
to inactivated whole ZEBOV virions (Makona strain)
as measured on ELISA. The data show that immuno
genicity at 4 weeks after priming with ChAd3 was simi
lar to that measured after immunization with a recombi
nant vesicular stomatitis virusbased vaccine expressing
ZEBOV glycoprotein (rVSV-ZEBOV) in 10 vaccinees in
Hamburg, Germany. Panel D shows titers of neutraliz
ing antibodies against live ZEBOV (Mayinga strain) from
all participants who received the MVA booster, as mea
sured at 28 days after the ChAd3 dose and 14 days after
the MVA dose. Low levels of neutralizing antibodies
that were detected in participants at 28 days were sim
ilar to levels reported after the administration of the
rVSV-ZEBOV vaccine. By 14 days after MVA vaccination,
the levels had increased by a factor of 9. In Panels C
and D, the columns represent the geometric mean titer,
the I bars represent 95% confidence intervals, and the
horizontal dashed lines represent the positive threshold.
In Panels B and D, the asterisk denotes P<0.001 by the
two-tailed MannWhitney test.
The dose of ChAd3 or MVA vaccine had no

significant effect on post-boost IgG titers, nor
did the interval between priming and boosting
vaccinations affect the magnitude of the antibody response (r=0.20, P=0.30) (Fig. S3C in the
Supplementary Appendix). However, there was

a significant positive correlation between the
primeboost interval and the neutralizing antibody titer, regardless of the MVA dose (r=0.72,
P<0.001) (Fig. S3D in the Supplementary Appendix). Antibody induction to the Sudan Ebola
virus glycoprotein was assessed, but as expected in the absence of priming with this antigen,
antibody titers against the Sudan virus glycoprotein were not detected after administration of the
prime vaccine, which suggests a lack of crossreactivity to the Sudan strain with antibodies
raised against the ZEBOV glycoprotein. However,
after boosting with the MVA vaccine (which expresses a Sudan Ebola virus glycoprotein), IgG
titers increased significantly (geometric mean
titer before boosting, 0.1; 95% CI, 0.07 to 0.2;
geometric mean titer 14 days after boosting, 1.5;
95% CI, 1.1 to 2.0; P<0.001 by the Friedman test)
(Fig. S3F in the Supplementary Appendix).
Cell-Mediated Immunity Induced
by Vaccination
ELISPOT responses peaked 7 days after boosting

with MVA at a median of 2068 spot-forming
cells (SFCs) (interquartile range, 1197 to 3447)
per million PBMCs and were significantly higher
than peak responses after prime vaccination at
14 days (SFCs, 633; interquartile range, 274 to 820;
P<0.001 by the two-tailed Wilcoxon matchedpairs test). Responses were maintained at 180
days after boosting (SFCs, 498; interquartile
range, 207 to 905) and were significantly higher
than non-boosted responses (SFCs, 84; interquartile range, 50 to 192; P<0.001 by the Mann
Whitney test) (Fig.2A). There was a modest
negative correlation between the primeboost
interval and the peak ELISPOT response
(r=0.42, P=0.03 by two-tailed Pearsons correlation coefficient) (Fig.2B). However, there was
no significant relationship between the magnitude of the antibody response and the T-cell response (Spearmans correlation coefficient, 0.17;
P=0.39).
Intracellular cytokine staining revealed that
all participants had positive CD4+ and CD8+
interferon- T-cell responses after boosting. The
median frequency of CD4+ T cells secreting
interferon-, interleukin-2, or tumor necrosis factor (TNF-) increased from 0.13% (interquartile range, 0.004 to 0.19) at 14 days after prime
vaccination to 0.20% (interquartile range, 0.15

1641
The
MVA 3108 PFU

MVA 1.5108 PFU
Unboosted
MVA
2000
1000
6000
4000
2000
21
28
35
+9
0
18
M 0
+1
80
90
+2
+0
+7
28
14
r=0.42
P=0.03
8000
CD4+
56
63
70
77
D Expression of Marker CD107a
CD8+
101
CD107a Expression
(% of CD8+ T-cell subset)
100
101
102
101
102
0
10
3.
rim
-p
Po
st
3.
0
10
5
10
1.
e
rim
-p
st
Po
3.
0
10
5
10
1.
rim
-p
Po
st
103
e
103
100
5
10
Total Cytokine Response

(% of subset)
49
PrimeBoost Interval (day)
Day
C Total Cytokine Response on Flow Cytometry
42
1.
SFCs per 106 PBMC
ChAd3
m e dic i n e
B PrimeBoost Interval and Peak ELISPOT Response
3000
SFCs per 106 PBMC (7 days after MVA)
A ELISPOT Response to MVA Boosting
of
MVA Dose (PFU)
MVA Dose (PFU)
E Cytokine Induction in CD4+ and CD8+ T Cells after MVA

CD4+ T Cells
1642
CD8+ T Cells
MVA: 1.5108 PFU
MVA: 1.5108 PFU
MVA: 3.0108 PFU
MVA: 3.0108 PFU
Interferon- Interleukin-2 TNF-

+

Figure 2 (facing page). T-Cell Responses and Induction

of Cytokines after Boosting with MVA.
Panel A shows the median T-cell responses to ChAd3
vaccination and MVA boosting on enzyme-linked im
munosorbent spot (ELISPOT) assay at all time points,
as measured in spot-forming cells (SFCs) per million
peripheral-blood mononuclear cells (PBMCs). The dose
of MVA is indicated in plaque-forming units (PFU).
Panel B shows the relationship between the prime
boost interval and the peak ELISPOT response 7 days
after MVA vaccination, as calculated by means of a
two-tailed Spearmans test. Panel C shows the total
c ytokine response on flow cytometry with intracellular
cytokine staining at 28 days after priming (post-prime)
or 7 days after boosting, according to the MVA dose.
The secretion of interferon-, interleukin-2, and tumor
necrosis factor (TNF-) by CD4+ and CD8+ T cells
was quantified for each booster-dose group and ex
pressed as the frequency of cells expressing any one
of the three cytokines. Panel D shows the expression
of the degranulation marker CD107a 28 days after prim
ing or 7 days after boosting. In Panels C and D, the
solid horizontal lines indicate median values, and the
dashed horizontal lines indicate the positive threshold.
Panel E shows the proportions of CD4+ and CD8+ T cells
that secreted any combination of interferon-, inter
leukin-2, and TNF- after stimulation with two differ
ent MVA doses.
Short-Interval Boosting
Given the pivotal role that has been shown for

T cells in preclinical efficacy studies in macaques
and the finding of high T-cell and antibody
immunogenicity even with the shortest prime
boost intervals, we assessed the effect of re
ducing the primeboost interval further to either
1 week or 2 weeks in two groups of eight participants each. ELISPOT responses in the two
groups still peaked at 7 days after boosting
(Fig.3A). We observed a modest negative correlation between the primeboost interval and peak
T-cell immunogenicity among all participants
(r=0.30, P=0.04 by two-tailed Spearmans correlation coefficient) (Fig.3B). In a comparison of
the median ELISPOT response in groups that
received the MVA booster dose at an interval of
1 week, 2 weeks, or 3 to 10 weeks after the priming vaccination, there were no significant betweengroup differences at either 7 days or 28 days
after the MVA dose (Fig.3C and 3D).
An analysis of antibody responses showed
that reducing the primeboost interval resulted
in a decrease in the peak IgG titer after the MVA
dose (P<0.05 for all comparisons) (Fig. S4A in
the Supplementary Appendix). Additional celluto 0.31) at 7 days after MVA boosting (P<0.001 lar and humoral immunologic analyses are deby the KruskalWallis test) (Fig.2C). The increase scribed in the Supplementary Appendix.
in the median frequency of cytokine-secreting
CD8+ T cells was even more pronounced, from
Discussion
0.004% (interquartile range, 0.004 to 0.09) at 14
days after prime vaccination to 0.25% (interquar- The boosting ability of MVA, particularly to entile range, 0.10 to 0.65) at 7 days after MVA boost- hance T-cell responses, has been described preing (P<0.001 by the KruskalWallis test).
clinically and clinically for vaccine candidates
The expression of the degranulation marker targeting several diseases18-22; however, data on
CD107a on CD8+ T cells increased by a factor of boosting of virus-specific human neutralizing
5 after boosting (P=0.003 by the KruskalWallis antibodies are lacking. In our study, we assessed
test) (Fig.2D). The MVA dose had no significant such boosting ability with respect to the ChAd3
effect on the magnitude of the T-cell response as vaccine, a relatively immunogenic priming agent,
measured by means of ELISPOT (Fig.2A) or in- and report large enhancements of antibody and
tracellular cytokine staining (Fig.2C and 2D). T-cell immunogenicity. We found induction of
Analysis of polyfunctionality confirmed the human neutralizing antibodies to Ebola virus at
dominance of TNF-secreting CD4+ T cells substantial titers by boosting, which correlated
over cells secreting either interferon- or inter- with the overall increased magnitude of antileukin-2 (Fig.2E). Cells that were positive only body titers on IgG ELISA assays. Antibodies to
for interferon- and double-positive cells secret- the Sudan strain of Ebola virus glycoprotein
ing interferon- and TNF- (with the latter being were also detected after boosting, albeit at a low
associated with protection in macaques17) were level. The induction of a response to the Sudan
the largest subgroups in the CD8+ T-cell re- strain is an important consideration for future
sponse (Fig.2E).
outbreak control. We also found an acceptable

1643
The
A ELISPOT with Reduced PrimeBoost Intervals
B ELISPOT According to PrimeBoost Interval
MVA
2 Wk interval
2000
m e dic i n e
10,000
1 Wk interval
SFCs per 106 PBMC
SFCs per 106 PBMC
3000
of
310 Wk interval
1000
r=0.30
P=0.04
8,000
6,000
4,000
2,000
0
14
D
0
21
28
35
42
49
56
63
70
77
PrimeBoost Interval (days)
D
0
(3
wk
)
(2
D wk)
0
(1
wk
)
M
+0
M
+7
M
+1
4
M
+2
8
Day
C ELISPOT 7 Days after MVA
D ELISPOT 28 Days after MVA

10,000
SFCs per 106 PBMC
SFCs per 106 PBMC
10,000
8,000
6,000
4,000
2,000
0
8,000
6,000
4,000
2,000
0
310
PrimeBoost Interval (wk)
310
PrimeBoost Interval (wk)
Figure 3. Effect of Reduced PrimeBoost Intervals on Cellular Immunogenicity.

Panel A shows the results of reducing the interval between prime vaccination with ChAd3 and booster vaccination
with MVA to either 1 week or 2 weeks (as compared with 3 to 10 weeks) in two groups of eight participants each.
The results are shown as median T-cell responses on ELISPOT assay, as measured in SFCs per million PBMCs. D0
indicates the beginning of the primeboost interval for each group. Responses in all three groups peaked at 7 days
after boosting, regardless of the primeboost interval. Panel B shows the relationship between the primeboost
interval and the peak ELISPOT response 7 days after boosting, with a modest negative correlation between the
primeboost interval and peak T-cell immunogenicity. Also shown are individual ELISPOT responses to summed
glycoprotein peptide pools at 7 days (Panel C) and 28 days (Panel D) after boosting; no significant differences be
tween the groups were seen at either 7 days or 28 days. The black horizontal lines indicate median values. In these
analyses, all the participants received 2.51010 viral particles of the ChAd3 vaccine and a booster dose of 1.510 8
PFU of MVA.
safety profile for MVA at the two doses and at

all intervals that we evaluated. We found that
boosting can be immunogenic for antibodies
and T cells at primeboost intervals as short as
1 week. Such short-interval regimens may facil
itate vaccine deployment in outbreak settings
where both rapid onset and durable vaccine efficacy are required.
A single dose of the ChAd3 vaccine induced
uniform protection shortly after vaccination and
partial longer-term protection in macaques.11 In
humans, the ChAd3 vaccine induced levels of
1644
anti-ZEBOV IgG and virus-neutralizing antibodies that were similar to the levels in the rVSVZEBOV ring vaccination study. Since no evidence
of cellular immunogenicity has yet been reported for the rVSV-ZEBOV vaccine, these vectors
probably induce different immune responses. The
induction of more CD4+ T cells than CD8+ T cells
after the administration of the ChAd3 vaccine
was unexpected on the basis of preclinical studies of these vaccine vectors, but we found that
the T-cell balance was reversed to greater levels
of CD8+ T cells after MVA boosting in humans.

This increase in CD8+ T-cell levels may enhance

the vaccine efficacy, since CD8+ T-cell depletion
was found to reduce the efficacy of an adeno
virus vaccine in macaques.13 The durability in
protection that we observed with this regimen
may result from help provided by CD4+ T cells.
The cellular immunogenicity induced by the
ChAd3 vaccine provides an additional potential
mechanism to provide greater vaccine efficacy
and durability than that provided by the rVSVZEBOV vaccine, although this hypothesis cannot
be confirmed without an efficacy trial.
The ChAd3 and MVA viral vectors have a number of other practical advantages in that largescale manufacturing processes concordant with
Good Manufacturing Practice standards have
been established and both vectors have been assessed in large numbers of vaccinees for a range
of indications without reports of any substantial
safety concerns to date.18,23-30 Nonreplicating viral
vectors have shown a reasonable safety profile
and may be preferred to replication-competent
vectors for widespread use in populations at risk
for undetected immunodeficiencies.
We also found antibody responses that remained positive 6 months after vaccination above
a threshold associated with efficacy in humans.
High-level durable efficacy is desirable for protecting populations against future epidemics and
may be particularly important for high-risk
populations such as health care workers. Singledose vaccines may prove to be preferable for logistic simplicity if just short-term efficacy is required in outbreak settings. However, we found
that a 1-week interval between the administration of the prime vaccine and the booster vaccine
provided CD8+ T-cell immunogenicity just 2 weeks

after the prime dose. We also found higher antibody responses than with single-dose vaccination,
even though such responses were lower than with
longer primeboost intervals. Taken together,
these data provide a basis for consideration of
particular vectored vaccine regimens for use in
either prevention or control of an outbreak.
The views expressed in this article are those of the authors
and do not necessarily represent the position or policies of the
World Health Organization (WHO).
Supported by the Wellcome Trust, the United Kingdom Medical Research Council, the United Kingdom Department for International Development, and the United Kingdom National Institute for Health Research Oxford Biomedical Research Centre.
The National Health Service Blood and Transplant and Public
Health England provided funding for the competition ELISA.
The ChAd3 vaccine was provided by the Vaccine Research Center of
the National Institute of Allergy and Infectious Diseases (NIAID)
and GlaxoSmithKline. MVA-BN Filo was produced under a contract (FBS-004-009) between the NIAID and Fisher BioServices
and a contract (HHSN272200800044C) between the National
Institutes of Health and Fisher Bioservices.
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org.
We thank the members of the data and safety monitoring
board: Peter Smith (chair), George Griffin, Saul Faust, Brian
Angus, Kwadwo Koram, Kalifa Bojang, and Mahamodou Thera;
the Wellcome Trust award scientific advisory board: Brian
Greenwood (chair), Peter Piot, Pontiano Kaleebu, Allan Saul,
Paul Kaye, Charlie Weller, and Morven Roberts; John Mascola of
the National Institutes of Health, Marie-Paul Kieny of the WHO,
Samba Sow of the Center for Vaccine Development of Mali, and
Umberto Dalessandro of the Medical Research Council of Gambia for their advice; Carly Banner and Geoff Lees for arranging
contracts; Xiangguo Qiu and Gary Kobinger of the Special
Pathogens Program, National Microbiology Laboratory, Public
Health Agency of Canada, for provision of anti-4G7 purified IgG
used in the competition ELISA; Steve Dicks and Lisa Hill for
technical assistance with the competition ELISA; Michael
Schmidt and Gotthard Ludwig, University of Marburg, for technical support in biosafety level 4; the Medicines and Healthcare
Products Regulatory Agency and the Oxfordshire research ethics
committee for exceptionally rapid review; and Vasee Moorthy for
serving as the study WHO liaison.
Appendix
The authors full names and academic degrees are as follows: Katie Ewer, Ph.D., Tommy Rampling, M.R.C.P., Navin Venkatraman,
M.R.C.P., Georgina Bowyer, B.A., Danny Wright, M.Sc., Teresa Lambe, Ph.D., EgeruanB. Imoukhuede, M.D., Ruth Payne, M.R.C.P.,
SarahKatharina Fehling, Ph.D., Thomas Strecker, Ph.D., Nadine Biedenkopf, Ph.D., Verena Krhling, Ph.D., ClaireM. Tully, B.A.,
NickJ. Edwards, B.Sc., EmmaM. Bentley, B.Sc., Dhanraj Samuel, Ph.D., Genevive Labb, Ph.D., Jing Jin, Ph.D., Malick Gibani,
M.R.C.P., Alice Minhinnick, M.B., Ch.B., Morven Wilkie, M.R.C.P., Ian Poulton, Dip.H.E., Natalie Lella, B.A., Rachel Roberts, M.Sc.,
Felicity Hartnell, M.B., B.S., Carly Bliss, B.A., Kailan SierraDavidson, B.A., Jonathan Powlson, B.Sc., Eleanor Berrie, Ph.D., Richard
Tedder, M.B., B.Chir., Francois Roman, M.D., Iris DeRyck, Ph.D., Alfredo Nicosia, Ph.D., NancyJ. Sullivan, Ph.D., DaphneA. Stanley,
M.S., OlivierT. Mbaya, M.D., JulieE. Ledgerwood, D.O., RichardM. Schwartz, Ph.D., Loredana Siani, Ph.D., Stefano Colloca, Ph.D.,
Antonella Folgori, Ph.D., Stefania DiMarco, Ph.D., Riccardo Cortese, M.D., Edward Wright, Ph.D., Stephan Becker, Ph.D., BarneyS.
Graham, M.D., RichardA. Koup, M.D., MyronM. Levine, M.D., Ariane Volkmann, Ph.D., Paul Chaplin, Ph.D., AndrewJ. Pollard, Ph.D.,
SimonJ. Draper, D.Phil., W.Ripley Ballou, M.D., Alison Lawrie, Ph.D., SarahC. Gilbert, Ph.D., and AdrianV.S. Hill, D.M.
The authors affiliations are as follows: the Jenner Institute and Centre for Clinical Vaccinology and Tropical Medicine, University of
Oxford, and the National Institute for Health Research Oxford Biomedical Research Centre, Oxford (K.E., T.R., N.V., G.B., D.W., T.L.,
E.B.I., R.P., C.M.T., N.J.E., G.L., J.J., M.G., A.M., M.W., I.P., N.L., R.R., F.H., C.B., K.S.-D., J.P., E.B., A.J.P., S.J.D., A.L., S.C.G.,
A.V.S.H.), and Viral Pseudotype Unit, Faculty of Science and Technology, University of Westminster (E.M.B., E.W.), and Virus Reference
Department, Public Health Agency (D.S., R.T.), London all in the United Kingdom; the Institute of Virology, Philipps University
Marburg (S.K.F., T.S., N.B., V.K., S.B.), and German Center for Infection Research, Partner Site GiessenMarburgLangen (S.B.),

1645
Marburg, and Bavarian Nordic, Martinsried (A.V., P.C.) all in Germany; GlaxoSmithKline Biologicals, Rixensart, Belgium (F.R.,
I.D.R., W.R.B.); ReiThera, Rome (A.N., L.S., S.C., A.F., S.D.M.), and CEINGE and the Department of Molecular Medicine and Medical
Biotechnology, University of Naples Federico II, Naples (A.N.) both in Italy; Vaccine Research Center, National Institute of Allergy
and Infectious Diseases, National Institutes of Health, Bethesda (N.J.S., D.A.S., O.T.M., J.E.L., R.M.S., B.S.G., R.A.K.), and the Center
for Vaccine Development, University of Maryland School of Medicine, Baltimore (M.M.L.) both in Maryland; and Keires, Basel,
Switzerland (R.C.).
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The

A Monovalent Chimpanzee Adenovirus

In this phase 1 study, we administered a single dose of the chimpanzee adenovirus 3

The authors full names, academic de

The New England Journal of Medicine

he recent outbreak of Ebola virus

studies with responses observed in rVSV-ZEBOV

The study was conducted at the Centre for

n engl j med 374;17nejm.org April 28, 2016

The New England Journal of Medicine

n engl j med 374;17nejm.org April 28, 2016

The New England Journal of Medicine

Distribution no. (%)

Race no. (%)

Distribution no. (%)

Sex no. (%)

Table 1. Characteristics of the Participants at Baseline.*

A Monovalent Chimpanzee Adenovirus Ebola Vaccine

The study was reviewed and approved by the

In this phase 1 study, we administered the ChAd3

design are provided in the study protocol, also

A total of 76 of the 123 volunteers who were

n engl j med 374;17nejm.org April 28, 2016

The New England Journal of Medicine

A Monovalent Chimpanzee Adenovirus Ebola Vaccine

on day 1 after vaccination owing to an aversion

A complete list of the frequency and maximum

Antibody responses as measured by means of

n engl j med 374;17nejm.org April 28, 2016

The New England Journal of Medicine

A Antibody Response on Standardized ELISA

B Antibody Response on ZEBOV GP IgG ELISA

Standardized ZEBOV GP IgG

2.51010 Viral particles

51010 Viral particles

C Antibody Response in Whole ZEBOV Virions

11010 Viral particles

D Neutralizing Antibodies against Live ZEBOV

Vaccine Regimen (day)

Neutralizing Antibody Titer

n engl j med 374;17nejm.org April 28, 2016

The New England Journal of Medicine

A Monovalent Chimpanzee Adenovirus Ebola Vaccine

Figure 1 (facing page). Antibody Responses to the Zaire

The dose of ChAd3 or MVA vaccine had no

Supplementary Appendix). However, there was

ELISPOT responses peaked 7 days after boosting

n engl j med 374;17nejm.org April 28, 2016

The New England Journal of Medicine

MVA 3108 PFU

D Expression of Marker CD107a

Total Cytokine Response

PrimeBoost Interval (day)

C Total Cytokine Response on Flow Cytometry

SFCs per 106 PBMC

B PrimeBoost Interval and Peak ELISPOT Response

SFCs per 106 PBMC (7 days after MVA)

A ELISPOT Response to MVA Boosting

MVA Dose (PFU)

MVA Dose (PFU)

E Cytokine Induction in CD4+ and CD8+ T Cells after MVA

MVA: 1.5108 PFU

MVA: 1.5108 PFU